The mechanisms responsible for pathfinding are largely unexplored. Pathfinding is defined in two-dimensional culture as the formation of ventral protrusions that are required for focal contact formation. The reason that the relationships between ventral protrusions, lateral protrusions and adhesion formation have not been adequately studied to date is in part due to the inability of current light microscopes to image actin polymerization dynamics at the ventral surface in live cells during stimulated protrusion. In this application we describe how recent advances in optical imaging may be combined with existing biosensors in order to elucidate the choreographed sequence of spatial and temporal events that are involved in ventral protrusions and their relationship to focal contact formation. We will: 1. Extend imaging methods for observation of ventral cell surface dynamics to live cells. a) Develop hardware and software to enable total internal reflection fluorescence (TIRF) imaging using excitation from a pulsed laser. b) Develop hardware and software to enable fluorescence lifetime imaging microscopy (FLIM) using TIRF illumination. c) Develop hardware and software in order to optimize interference microscopy (IRM). 2. Visualize ventral protrusions in live cells and determine the actin compartments on the ventral surface that contribute to ventral protrusion activity and the formation of local contacts. [unreadable] [unreadable]